PROJECT SUMMARY Mixed lineage leukemia is an aggressive subset of acute leukemias with poor clinical outcome and a severe need for improved treatment options. A genetic hallmark of mixed lineage leukemia is chromosomal translocation of the MLL gene, resulting in the formation of MLL-fusion proteins that drive leukemogenesis by mislocalizing essential cellular epigenetic machinery. Many MLL-fusion partner proteins interact with the histone methyltransferase Dot1L, mistargeting its activating methylation mark to developmental regulatory genes that are native targets of MLL during development. Dot1L catalytic activity is essential for both leukemogenic transformation and maintenance, making Dot1L inhibition one of the major strategies underlying current therapeutic development. Defining the molecular basis of Dot1L activity is an essential step towards rational development of Dot1L-targeted therapeutics, but our understanding of the structural and mechanistic basis for Dot1L activity on its native nucleosome substrate is still incomplete. Dot1L methylation of its target histone H3 lysine 79 residue is dependent on prior ubiquitylation of its nucleosome substrate on histone H2B at lysine 120. However, the structural basis for this trans-histone crosstalk has not been established. To elucidate the mechanistic basis for Dot1L activity on its nucleosome substrate and identify novel strategies for Dot1L inhibition, we solved a 3.9 cryo-EM structure of Dot1L bound to a site-specifically ubiquitylated nucleosome, providing the first high-resolution insight into how Dot1L engages with its nucleosome substrate and is regulated by ubiquitin. Guided by this structure, we identified residues in Dot1L essential for both its nucleosome-specific activity and upregulation by ubiquitin. In Aim 1, we will mutate these Dot1L residues to observe their effect on leukemia cell viability and Dot1L activity at known oncogenes. This work will define the nucleosome-specific and ubiquitin-dependent activities of Dot1L in leukemogenesis and probe the potential of the Dot1L-nucleosome and Dot1L-ubiquitin interfaces as therapeutic targets. Many MLL-fusion partner proteins are also components of the super elongation complex (SEC), a large transcriptional regulatory protein complex that promotes the elongation phase of transcription by releasing RNA polymerase II from promoter-proximal pausing. The SEC is also essential for leukemogenic transformation in mixed lineage leukemia, further promoting misregulation of gene expression at MLL-fusion target genes through overactivation of transcriptional elongation. Still, the structural organization of the SEC and the influence of MLL- fusions on SEC structure and function has not been explored. In Aim 2, I propose a complete structural and functional characterization of the SEC to determine the mechanistic basis for SEC-driven leukemogenic progression in the context of MLL-fusion proteins. Together, these aims will identify novel targets along the molecular pathways driving leukemogenesis to form the pre-clinical foundation for rational therapeutic development.